A method for preparing core-shell microcapsules based on a microfluidic chip

Abstract

The application discloses a preparation method of core-shell microcapsules based on a microfluidic chip and belongs to the technical field of microcapsule production. The method is simple and easy to implement, and can obtain microcapsules with excellent monodispersity, controllable size and pH response characteristics. The core-shell microcapsules can be applied to the field of probiotic packaging and delivery, and can significantly improve the survival rate of probiotics in harsh environments. The core-shell microcapsules also have pH response characteristics, can maintain the original shape under the acidic pH of the stomach, and can crack and release probiotics under the neutral pH of the intestinal tract, thereby achieving good gastrointestinal sustained-release function.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microcapsule production, and particularly relates to a preparation method of core-shell microcapsules based on a microfluidic chip. BACKGROUND

[0002] Probiotics have many health benefits, such as regulating intestinal flora balance, reducing serum cholesterol levels and the risk of cardiovascular disease. This makes the food industry increasingly demand functional foods containing probiotics. So far, probiotics are usually added to dairy matrices, but dairy products are often not a good choice for consumers who are allergic to dairy products and have lactose intolerance. Compared with dairy products, fruit juice has a different flavor and contains a variety of nutrients, so it can be used as a potential food matrix for probiotics. However, the acidic pH of fruit juice can affect the shelf stability of probiotics during the shelf life. In addition, probiotics are also vulnerable to the harsh environment of the human gastrointestinal tract during delivery to the intestine. Therefore, effective strategies need to be developed to improve the viability of probiotics. Among them, microencapsulation technology, as one of the most promising strategies, has been widely used to improve the survival rate of probiotics in harsh environments such as high temperature, oxygen and extremely low pH.

[0003] Some previous studies have successfully prepared core-shell microcapsules with hydrophobic materials (such as shellac, oil, etc.) as the core by the method of coaxial co-extrusion. However, the size of the microcapsules prepared by this co-extrusion method is usually greater than 1 mm, and the particle size distribution is wide. Generally speaking, smaller microcapsules are more favored because they do not affect the taste of food and allow greater molecular diffusion. Microfluidic technology is a technology that involves manipulating fluids using micron-scale channels, which provides a powerful method to quickly produce micron-sized monodisperse droplets. In addition, by designing the channels of the microfluidic system, probiotics can be more accurately incorporated into the droplets, and other materials can also be incorporated to better protect them in harsh environments. Unlike the external gelation method of co-extrusion by dropping sodium alginate droplets into a gel bath, microfluidic technology can produce sodium alginate microcapsules by internal gelation. This method is to disperse insoluble calcium complexes (such as CaCO3, EDTA-Ca) in a sodium alginate solution, and when the pH decreases, calcium ions are released from the complex and crosslink with sodium alginate to form a uniform hydrogel. Because the gelation process is from the inside out, it greatly guarantees the integrity of the droplets after solidification. SUMMARY

[0004] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and provide a preparation method of core-shell microcapsules based on a microfluidic chip, which is simple and easy to operate and can obtain microcapsules with excellent monodispersity, controllable size and pH response characteristics. The core-shell microcapsules can be applied to the field of probiotic encapsulation and delivery, and significantly improve the survival rate in harsh environments.

[0005] The present application provides a preparation method of core-shell microcapsules, comprising the following preparation steps:

[0006] (1) Preparation of hydrogel precursor solution: mix CaCl2 solution and Na2-EDTA solution and adjust the pH to neutral with NaOH aqueous solution, then stir, add sodium alginate and mix uniformly to prepare the hydrogel precursor solution;

[0007] (2) Preparation of core-shell microcapsule core layer material: stir the protein in water to prepare a protein solution, mix the protein solution with the sodium alginate solution to prepare a mixed solution, then adjust the pH of the mixed solution to neutral, and then add vegetable oil to the mixed solution and homogenize to obtain the core-shell microcapsule core layer material;

[0008] (3) Preparation of core-shell microcapsule shell layer material: dissolve nanocellulose in water and stir to obtain a nanocellulose solution, then mix the kelp nanocellulose solution with the hydrogel precursor solution prepared in step (1) to obtain the core-shell microcapsule shell layer material;

[0009] (4) Production of core-shell microcapsules: mix Tween 80, acetic acid and vegetable oil to prepare an external oil phase, then pump the core-shell microcapsule core layer material prepared in step (3) as an internal phase and the core-shell microcapsule shell layer material prepared in step (4) as an intermediate phase into a microfluidic chip, perform droplet formation of the internal gel in the chip, then collect in a CaCl2 solution, and then centrifuge to obtain the core-shell microcapsules.

[0010] Further, the concentration of the CaCl2 solution in step (1) is 50-120 mM.

[0011] Further, the concentration of the Na2-EDTA solution in step (1) is 50-120 mM.

[0012] Further, the volume ratio of the CaCl2 solution to the Na2-EDTA solution in step (1) is 1:0.8-1.2.

[0013] Further, the concentration of the NaOH aqueous solution in step (1) is 0.8-1.2 mol / L.

[0014] Further, the mass fraction of sodium alginate in the hydrogel precursor solution in step (1) is 0.5-4%.

[0015] Further, the protein in step (2) includes edible fish by-product protein, edible fish protein, and other commercially available proteins that can be used for Pickering stabilization.

[0016] Specifically, the protein in step (2) includes one or more of salmon protein and cod protein.

[0017] Further, the protein in the protein solution in step (2) has a mass concentration of 1-5%.

[0018] Further, the sodium alginate in the sodium alginate solution in step (2) has a mass concentration of 0.5-4%.

[0019] Further, the volume ratio of the protein solution to the sodium alginate solution in step (2) is 1:0.8-1.2.

[0020] Further, the homogenization in step (2) is performed at a speed of 7000-8000 rpm for 20-30 s.

[0021] Further, the plant oil in step (2) is one or more of corn oil, soybean oil, rapeseed oil, peanut oil, algal oil, and palm oil.

[0022] Further, the volume ratio of the plant oil to the mixed solution in step (2) is 1:0.8-1.2.

[0023] Further, the homogenization after adding the oil in step (2) is performed at a speed of 8000-15000 rpm for 0.5-3 min.

[0024] Specifically, the nanocellulose in step (3) is kelp nanocellulose.

[0025] Further, the nanocellulose in the nanocellulose solution in step (3) has a mass concentration of 0.1-0.5%.

[0026] Further, the volume ratio of the kelp nanocellulose solution to the hydrogel precursor solution in step (3) is 1:0.8-1.2.

[0027] Further, the plant oil in step (4) is one or more of corn oil, soybean oil, rapeseed oil, peanut oil, algal oil, and palm oil.

[0028] Further, the volume ratio of Tween 80 in the external phase oil phase in step (4) is 0.5%-2%.

[0029] Further, the volume ratio of acetic acid in the external phase oil phase in step (4) is 0.02%-0.5%.

[0030] Further, the inner phase flow rate in step (4) is 200-700 μL / h.

[0031] Preferably, the inner phase flow rate in step (4) is 500 μL / h.

[0032] Further, the intermediate phase flow rate in step (4) is 200-900 μL / h.

[0033] Preferably, the intermediate phase flow rate in step (4) is 800 μL / h.

[0034] Further, the outer phase flow rate in step (4) is 3000-4000 μL / h.

[0035] Preferably, the outer phase flow rate in step (4) is 3600 μL / h.

[0036] Further, the centrifugation condition in step (4) is centrifugation at 2000-4000 rpm for 1-5 min.

[0037] Further, the preparation process of the microfluidic chip in step (4) comprises the following steps:

[0038] First, a mask for photolithography is prepared, then, photoresist SU-8 is uniformly coated on the surface of a clean silicon wafer, and is baked at 60℃ and 90℃ for 1 min and 5 min respectively, followed by UV exposure, to manufacture a positive photoresist pattern with microchannels, and then heat treatment at 90℃ for 5 min is performed again;

[0039] Then, according to a ratio of 10:1 (v / v), polydimethylsiloxane and a curing agent are mixed, the mixture is poured on the positive photoresist pattern, then vacuum treatment is performed for 30 min, and heating at 90℃ for 30 min is performed, after which cutting and punching are performed, to obtain a chip substrate with a microchannel structure;

[0040] Then, a cover sheet without microchannels is prepared by using the same method, the cover sheet and the chip substrate are combined by plasma sealing, and heat treatment at 65℃ for 30 min is performed, to obtain a microfluidic chip.

[0041] Further, the mass concentration of CaCl2 in the CaCl2 solution in step (4) is 1-3%.

[0042] The present application provides a core-shell microcapsule prepared according to the above method.

[0043] The present application provides the application of the method in the field of preparing core-shell microcapsules.

[0044] The present application provides a preparation method of a bacteria-loaded core-shell microcapsule, comprising the following steps:

[0045] (1) Preparation of hydrogel precursor solution: CaCl2 solution is mixed with Na2-EDTA solution and the pH is adjusted to neutral with NaOH aqueous solution, followed by stirring, then adding sodium alginate and mixing uniformly to prepare the hydrogel precursor solution;

[0046] (2) Preparation of Lactobacillus plantarum powder oil: the probiotic bacteria are inoculated in MRS broth and incubated at 35-38℃ for 15-18h to activate the probiotic bacteria. After two subcultures, the probiotic bacteria precipitate is collected by centrifugation. After washing twice with sterile normal saline, the probiotic bacteria precipitate is added with a freeze-drying protective solution, then freeze-dried to obtain probiotic bacteria powder, which is then added to vegetable oil to prepare probiotic bacteria vegetable oil;

[0047] (3) Preparation of core layer material of core-shell microcapsule: protein is stirred in water to prepare a protein solution, which is then mixed with sodium alginate solution to prepare a mixed solution. The pH of the mixed solution is then adjusted to neutral, and then the probiotic bacteria vegetable oil is added to the mixed solution and homogenized to obtain the core layer material of core-shell microcapsule;

[0048] (4) Preparation of shell layer material of core-shell microcapsule: nanocellulose is dissolved in water to obtain a nanocellulose solution, which is then mixed with the hydrogel precursor solution prepared in step (1) to obtain the shell layer material of core-shell microcapsule;

[0049] (5) Production of core-shell microcapsule: Tween 80, acetic acid and vegetable oil are mixed to prepare an external oil phase, then the core layer material of core-shell microcapsule prepared in step (3) is used as an internal phase, and the shell layer material of core-shell microcapsule prepared in step (4) is used as an intermediate phase, which are pumped into a microfluidic chip respectively, and the internal gel droplets are formed in the chip, then collected in CaCl2 solution, and then centrifuged to obtain the core-shell microcapsule.

[0050] Further, the concentration of CaCl2 solution in step (1) is 50-120mM.

[0051] Further, the concentration of Na2-EDTA solution in step (1) is 50-120mM.

[0052] Further, the volume ratio of CaCl2 solution to Na2-EDTA solution in step (1) is 1:0.8-1.2.

[0053] Further, the concentration of NaOH aqueous solution in step (1) is 0.8-1.2mol / L.

[0054] Further, the mass fraction of sodium alginate in the hydrogel precursor solution in step (1) is 0.5-4%.

[0055] Further, the probiotic bacteria in step (2) include, but are not limited to, one or more of Bifidobacterium, Paracasei, Casei, Plantarum, Rhamnosus, Streptococcus thermophilus, Fermentum, Helveticus.

[0056] Further, the freeze-drying protective solution in step (2) is a mixed aqueous solution of sugars and proteins.

[0057] Further, the sugars in step (2) include, but are not limited to, one or more of sucrose, inulin, trehalose, lactose.

[0058] Further, the mass concentration of the sugars in the freeze-drying protective solution in step (2) is 5-10%.

[0059] Further, the proteins in step (2) include, but are not limited to, one or more of protein peptone, whey protein, sodium caseinate, gelatin, skim milk powder.

[0060] Further, the mass concentration of the proteins in the freeze-drying protective solution in step (2) is 5-15%.

[0061] Preferably, the sugars are inulin.

[0062] Preferably, the sugar proteins are skim milk powder.

[0063] Preferably, the concentration of inulin in the protective agent solution is 5-10wt%, and the concentration of skim milk powder in the protective agent solution is 5-15wt%.

[0064] Further, the mass-volume ratio of the probiotic bacteria powder and the freeze-drying protective solution in step (2) is 1g:5-20mL.

[0065] Further, the mass-volume ratio of the probiotic bacteria powder and the vegetable oil in step (2) is 1g:10-30mL.

[0066] Further, the proteins in step (3) include edible fish by-product proteins, edible fish proteins, and other commercially available proteins for Pickering stabilization.

[0067] Specifically, the proteins in step (3) include one or more of salmon proteins and cod proteins.

[0068] Further, the mass concentration of the proteins in the protein solution in step (3) is 1-5%.

[0069] Further, the mass concentration of sodium alginate in the sodium alginate solution in step (3) is 0.5-4%.

[0070] Further, the volume ratio of the protein solution and the sodium alginate solution in step (3) is 1:0.8-1.2.

[0071] Further, the homogenization condition in step (3) is 7000-8000 rpm for 20-30 seconds.

[0072] Further, the plant oil in step (3) is one or more of corn oil, soybean oil, rapeseed oil, peanut oil, algae oil, and palm oil.

[0073] Further, the volume ratio of the plant oil and the mixed solution in step (3) is 1:0.8-1.2.

[0074] Further, the homogenization condition after adding the oil in step (3) is 8000-15000 rpm for 0.5-3 minutes.

[0075] Specifically, the nanocellulose in step (4) is kelp nanocellulose.

[0076] Further, the mass concentration of the nanocellulose in the nanocellulose solution in step (4) is 0.1-0.5%.

[0077] Further, the volume ratio of the kelp nanocellulose solution and the hydrogel precursor solution in step (4) is 1:0.8-1.2.

[0078] Further, the plant oil in step (5) is one or more of corn oil, soybean oil, rapeseed oil, peanut oil, algae oil, and palm oil.

[0079] Further, the volume ratio of Tween 80 in the external phase oil phase in step (5) is 0.5%-2%.

[0080] Further, the volume ratio of acetic acid in the external phase oil phase in step (5) is 0.02%-0.5%.

[0081] Further, the inner phase flow rate in step (5) is 200-700 μL / h.

[0082] Preferably, the inner phase flow rate in step (5) is 500 μL / h.

[0083] Further, the intermediate phase flow rate in step (5) is 200-900 μL / h.

[0084] Preferably, the intermediate phase flow rate in step (5) is 800 μL / h.

[0085] Further, the external phase flow rate in step (5) is 3000-4000 μL / h.

[0086] Preferably, the flow rate of the outer phase in step (5) is 3600 μL / h.

[0087] Further, the centrifugation condition in step (5) is centrifugation at 2000-4000 rpm for 1-5 min.

[0088] Further, the preparation process of the microfluidic chip in step (5) comprises the following steps:

[0089] First, a mask for photolithography is prepared, then, photoresist SU-8 is uniformly coated on the surface of a clean silicon wafer, and is baked at 60℃ and 90℃ for 1 min and 5 min respectively, followed by UV exposure, to manufacture a positive photoresist pattern with microchannels, and then heat treatment at 90℃ for 5 min is performed again;

[0090] Then, polydimethylsiloxane and a curing agent are mixed in a ratio of 10:1 (v / v), the mixture is poured on the positive photoresist pattern, then vacuum treatment is performed for 30 min, and heating at 90℃ for 30 min is performed, after which cutting and punching are performed, to obtain a chip substrate with a microchannel structure;

[0091] Then, a cover sheet without a microchannel is prepared by using the same method, the cover sheet and the chip substrate are combined by plasma sealing, and heat treatment at 65℃ for 30 min is performed, to obtain a microfluidic chip.

[0092] Further, the mass concentration of CaCl2 in the CaCl2 solution in step (5) is 1-3%.

[0093] The present application provides a bacteria-carrying core-shell microcapsule prepared according to the above method.

[0094] The bacteria-carrying core-shell microcapsule obtained by the present application has applications in the fields of food, medicine preparation, and health product preparation.

[0095] Further, the medicine and health product is a medicine and health product containing probiotics.

[0096] Compared with the prior art, the present application has the following beneficial effects:

[0097] (1) The present application uses a microfluidic chip to controllably, quickly and gently prepare core-shell microcapsules with good monodispersity and micron-level size.

[0098] (2) The present application encapsulates probiotics in the core emulsion of the core-shell microcapsule, effectively avoiding direct contact of the probiotics with the harsh environment outside, thereby significantly improving the survival rate of the probiotics during gastrointestinal digestion and juice storage.

[0099] (3) The core-shell microcapsule prepared by the application has pH response characteristics, can maintain the original shape under the acidic pH of the stomach, and is broken and releases probiotics under the neutral pH of the intestine, thereby achieving good gastrointestinal sustained-release function. BRIEF DESCRIPTION OF DRAWINGS

[0100] Figure 1 It is a schematic diagram of a PDMS microfluidic device for generating probiotic sodium alginate microcapsules and a process diagram of preparing core-shell microcapsules on the chip.

[0101] Figure 2 Preparation of core-shell microcapsules based on microfluidic chips. Only the bright field images of the outer phase (A), the middle phase (B), the inner phase (C), the inner phase and the middle phase (D) when they are connected to the microfluidic chip, and the process images of the core-shell microcapsules (E1-E4), single-shell microcapsules (F1-F4) and single-core microcapsules (G1-G4) are formed.

[0102] Figure 3 It is a bright field image of the core-shell microcapsule (A), single-shell microcapsule (B) and single-core microcapsule (C) after solidification by the inner and outer gel method. The scale bar represents 1000 μm.

[0103] Figure 4 It is the influence of different fluid parameters on the diameter and polydispersity of the microcapsules. The particle size distribution and size of the microcapsules under different outer phase flow rates (A) and the number of microcapsules prepared from 100 μL of the inner phase under different inner phase flow rates (B).

[0104] Figure 5 It is the viable cell count of the probiotic microcapsules during the simulated gastrointestinal digestion process (A) and the morphology at different time points during the simulated digestion (B).

[0105] Figure 6 It is the viable cell count of the probiotic microcapsules stored in fruit juice at 25℃ (A) and 4℃ (B) for four weeks. DETAILED DESCRIPTION

[0106] In order to better understand the technical problems, technical solutions and technical effects solved by the application, the content of the application will be further described in detail below in combination with the embodiments and the drawings, but should not be understood as a limitation on the application.

[0107] Source of raw materials

[0108] The source of Lactobacillus plantarum used in the following examples is: Lactobacillus plantarum Lp90 purchased from Zhenjiang Tianyi Biotechnology Co., Ltd.;

[0109] The preparation method of salmon by-product protein used in the following examples is prepared according to steps (1)-(3) of S1 in Example 1 of CN116349867A;

[0110] The preparation method of kelp nanocellulose used in the following examples is prepared according to the method disclosed in the specific implementation of CN202210480195.

[0111] The preparation of free probiotics is by inoculating Lactobacillus plantarum in MRS broth and incubating at 37°C for 16h to activate the probiotics. After two subcultures, the probiotic precipitate is collected by centrifugation at 8000rpm for 8min at 4°C.

[0112] In the following example description, wt% refers to the mass percentage, for example "2wt% sodium alginate" means that 100mL of aqueous solution contains 2g of sodium alginate.

[0113] Measurement method

[0114] (I) To quantify the effect of external phase flow rate on the properties of microcapsules. After collecting the microcapsules, take pictures by optical microscope and upload to imageJ software. The size of 100 microcapsules in each treatment group is measured and averaged, and their column scatter plot is made to observe their particle size distribution range. By checking the number of microcapsules prepared from 100μl of internal phase at different internal phase flow rates, the effect of internal phase flow rate on the properties of microcapsules is quantified.

[0115] (II) Method for determining the activity of microencapsulated probiotics in simulated gastric juice and intestinal juice: 1g pepsin and 0.9g NaCl are contained in each 100ml of simulated gastric juice, and the pH value is adjusted to 1.2 with 1.0M HCl. 1g of trypsin, 0.68g of KH2PO4 and 1g of bile salt are contained in each 100ml of simulated intestinal juice, and the pH value is adjusted to 7.0 with 1.0M NaOH. Both the simulated gastric juice and the simulated intestinal juice are sterilized by filtering through a 0.22μm membrane and preheated in a water bath at 37°C before use. The core-shell microcapsules prepared from 1mL of internal phase or single-core microcapsules or single-shell microcapsules prepared from 1mL of intermediate phase are mixed with 10mL of simulated gastric juice and stirred in a water bath at 37°C for 120min. After the end of the simulated gastric juice, the pH of the simulated gastric juice is immediately adjusted to 7.0, and 10mL of simulated intestinal juice is added. The microcapsules continue to be stirred in a water bath at 37°C at a speed of 150rpm for 360min. The morphology of the microcapsules is photographed at 0min, 120min of simulated gastric juice digestion and 10min, 30min, 60min, 120min, 180min and 360min of simulated intestinal juice digestion, respectively. After the end of in vitro simulated digestion, 10mL of PBS is added to the digestion solution and homogenized for 5min to release all the probiotics. The released probiotics are diluted with physiological saline and placed on MRS agar medium, and cultured in a 37°C incubator for 48h and counted, and the results are expressed as log CFU / g. Free probiotics are also studied as a control group.

[0116] (III) Viability of microencapsulated probiotics in fruit juice after 4 weeks of storage: The core-shell microcapsules prepared from 1 mL of the internal phase or the single-core microcapsules prepared from 1 mL of the intermediate phase or the single-shell microcapsules prepared from 1 mL of the intermediate phase were added to 20 mL of commercial pasteurized orange juice, respectively. After 1, 2, 3, and 4 weeks of storage at 4 °C and 25 °C, they were mixed with 20 mL of PBS (pH = 7.4) and then homogenized for 5 min using a high-speed homogenizer to lyse the microcapsules. The released probiotics were serially diluted with physiological saline and plated on MRS agar medium. The colonies were counted after 48 h of incubation at 37 °C and the results were expressed as log CFU / g. Free probiotics were also studied as a control group.

[0117] Example 1

[0118] This example is the process of preparing core-shell microcapsules.

[0119] (1) Preparation of microfluidic chip: First, the microchannel structure of the microfluidic chip was drawn using CAD software, and then the design pattern was made into a mask suitable for photolithography. The specific chip design pattern can be found in Figure 1 . Next, the photoresist SU-8 was uniformly coated on the surface of the cleaned silicon wafer using a spin coater, and was baked at 60 °C and 90 °C for 1 minute and 5 minutes, respectively, to ensure the uniformity and stability of the photoresist. Subsequently, the positive photoresist pattern with microchannels was manufactured using ultraviolet exposure technology, and was again heat treated at 90 °C for 5 minutes to ensure the stability of the structure. Then, according to the proportion of 10:1 (v / v), polydimethylsiloxane and curing agent were mixed, and after thorough mixing, the mixture was poured onto the positive photoresist pattern. Subsequently, a vacuum pump was used to extract the vacuum for 30 minutes to eliminate potential air bubbles, and the mixture was heated at 90 °C for 30 minutes to complete the curing process. Once the curing was completed, the chip substrate with microchannel structure was obtained at specific locations by cutting and punching. At the same time, the cover sheet without microchannels was prepared using the same method, and then the two were tightly combined by plasma sealing method. Finally, the packaged microfluidic chip was heat treated at 65 °C for 30 minutes to ensure that each part was stably and reliably connected.

[0120] (2) Preparation of homogeneous hydrogel precursor solution of EDTA-Ca complex and sodium alginate solution: The EDTA-Ca complex was prepared by mixing 100 mM CaCl2 solution and 100 mM Na2-EDTA solution at a ratio of 1:1 (v / v) and adjusting the pH to 7.2 using 1 mol / L NaOH aqueous solution and then stirring at 150 rpm for 1 h. The EDTA-Ca complex was uniformly mixed with sodium alginate to prepare a homogeneous hydrogel precursor solution containing calcium-EDTA complex (50 mM) and SA solution (2 wt%).

[0121] (3) Preparation of core material of core-shell microcapsule: Dissolve salmon protein powder in sterile water and stir to prepare a 4wt% salmon protein solution. Mix the 4wt% salmon protein solution with a 2wt% sodium alginate solution at a volume ratio of 1:1 and homogenize at a speed of 8000 rpm for 30 s to prepare a mixed solution. Then adjust the pH value of the mixed solution to 7, and then add corn oil into the mixed solution (the volume ratio of oil phase to water phase is 1:1, v / v), and then homogenize at a speed of 12000 rpm for 2 min to prepare a stable Pickering emulsion, i.e. the core material of the core-shell microcapsule.

[0122] (4) Preparation of shell material of core-shell microcapsule: Dissolve 0.02 g of kelp nanocellulose in 10 mL of deionized water and stir at 55°C for 3 h to obtain a 0.2wt% kelp nanocellulose solution. Then mix the kelp nanocellulose solution with 10 mL of hydrogel precursor solution and shake well to obtain the shell material of the core-shell microcapsule.

[0123] (5) Production of core-shell microcapsule on a chip: The core-shell microcapsule is prepared by an inside-out gel method on a microfluidic chip. Corn oil containing 1% (v / v) Tween 80 and containing 0.05% (v / v) acetic acid as an external phase is used to form core-shell droplets and induce the release of calcium ions. The core material of the core-shell microcapsule prepared in step (3) (inner phase), the shell material of the core-shell microcapsule prepared in step (4) (intermediate phase) and the external phase are respectively loaded into three 1 mL syringes and connected to a syringe pump. The inner phase, the intermediate phase and the external phase are pumped into the microfluidic chip at a flow rate of 500 μL / h, 800 μL / h and 3600 μL / h, respectively. The droplets of the inner gel are collected in a CaCl2(2wt%) solution in the chip, and the more stable core-shell microcapsule is obtained by the outer gel. Then centrifuge at 3000 rpm for 5 min to remove the excess oil phase to obtain the core-shell microcapsule, which is stored in a 4°C refrigerator for later use.

[0124] Comparative Example 1

[0125] This comparative example is a single-shell microcapsule prepared according to the preparation process of Example 1, except that step (3) is omitted, i.e. the inner phase in the preparation of the core-shell microcapsule is omitted, and the original step (5) is changed to the following operation:

[0126] Preparation of single-shell microcapsules on-chip: Single-shell microcapsules were prepared on a microfluidic chip by the inner-outer gelation method. Corn oil containing 1% (v / v) Tween 80 and corn oil containing 0.05% (v / v) acetic acid were used as the outer phase to form single-shell droplets and induce the release of calcium ions. The single-shell microcapsule shell material (middle phase) prepared from step (3) and the outer phase were loaded into two 1 mL syringes, respectively, and connected to the syringe pumps. The middle phase and the outer phase were pumped into the microfluidic chip at a flow rate of 800 μL / h and 3600 μL / h, respectively. The droplets with inner gelation were collected in a CaCl2(2 wt%) solution in the chip, and more stable single-shell microcapsules were obtained by outer gelation. After that, the single-shell microcapsules were centrifuged at 3000 rpm for 5 min to remove the excess oil phase, and stored in a 4 °C refrigerator for later use.

[0127] Comparative Example 2

[0128] This comparative example was to prepare single-core microcapsules. The preparation process referred to Example 1, only step (4) was omitted, i.e. the middle phase in the preparation of core-shell microcapsules was omitted, and the original step (5) was changed to the following operation:

[0129] Preparation of single-core microcapsules on-chip: Single-core microcapsules were prepared on a microfluidic chip by the inner-outer gelation method. Corn oil containing 1% (v / v) Tween 80 and corn oil containing 0.05% (v / v) acetic acid were used as the outer phase to form single-core droplets and induce the release of calcium ions. The single-core microcapsule core material (inner phase) prepared from step (3) and the outer phase were loaded into two 1 mL syringes, respectively, and connected to the syringe pumps. The inner phase and the outer phase were pumped into the microfluidic chip at a flow rate of 500 μL / h and 3600 μL / h, respectively. The droplets with inner gelation were collected in a CaCl2(2 wt%) solution in the chip, and more stable single-core microcapsules were obtained by outer gelation. After that, the single-core microcapsules were centrifuged at 3000 rpm for 5 min to remove the excess oil phase, and stored in a 4 °C refrigerator for later use.

[0130] Comparative Example 3

[0131] This comparative example was to prepare core-shell microcapsules. The preparation process referred to Example 1, only the flow rate of the outer phase fluid in step (5) was changed to 7200 μL / h.

[0132] Comparative Example 4

[0133] This comparative example was to prepare core-shell microcapsules. The preparation process referred to Example 1, only the flow rate of the outer phase fluid in step (5) was changed to 2400 μL / h.

[0134] Comparative Example 5

[0135] The core-shell microcapsules prepared in this comparative example were prepared by referring to the procedure of Example 1, except that the inner phase fluid flow rate in step (5) was changed to 200 μL / h.

[0136] Comparative Example 6

[0137] The core-shell microcapsules prepared in this comparative example were prepared by referring to the procedure of Example 1, except that the inner phase fluid flow rate in step (5) was changed to 600 μL / h.

[0138] The process of preparing the microcapsules on the chip in Example 1 and Comparative Examples 1 and 2 is shown in Figure 2 As shown in Figure 2 A, Figure 2 B), when only the outer phase or the intermediate phase was injected into the chip, there was no obvious phenomenon in the chip because sodium alginate and corn oil were transparent. Figure 2 C). At this time, when the intermediate phase was injected into the chip at a higher flow rate at the same time, the inner phase was squeezed by the intermediate phase to generate obvious coaxial fluid Figure 2 D). As shown in Figure 2 E, the coaxial fluid was cut by corn oil one by one at the second connection to generate core-shell microcapsules with good monodispersity (Example 1). The addition of Span 80, a surfactant, in the outer phase was used to stabilize and prevent the aggregation of droplets, thereby improving the monodispersity of the microcapsules. In addition, the single-shell microcapsules prepared by only the intermediate phase (Comparative Example 1) and the single-core microcapsules prepared by only the inner phase (Comparative Example 2) were completely transparent (Comparative Example 1) Figure 2 F, Figure 2 G) compared with the core-shell microcapsules.

[0139] The bright field images of the gelled microcapsules in Example 1 and Comparative Examples 1 and 2 are shown in Figure 3 A Figure 3 B and Figure 3 C, respectively. Under the appropriate fluid flow rate, the shape of the microcapsules after gelling was observed to be complete and uniform in size. Interestingly, the present application found that the shape of the core inside the core-shell microcapsules was irregular, which may be because the core solution inside was free-flowing before the droplet solidified. Secondly, because the shell layer contacted the corn oil containing acetic acid before the core, it solidified before the core, which made it difficult for the core and the shell layer to fuse together before solidification. After the complete solidification of the core, the calcium ions were released, and the shape of the core before solidification was retained. In summary, compared with Comparative Examples 1 and 2, the core-shell microcapsules with a multi-layer structure (Example 1) were successfully prepared by the chip method, and this multi-layer structure not only presented an interesting visual effect on the microscale, but also endowed the microcapsules with unique physical and chemical properties.

[0140] In addition, the size of the microcapsules not only affects the taste of the food, but also plays a key role in the encapsulation and protection of probiotics. The present application further discusses the key parameters that affect the characteristics of the microcapsules, including the flow rates of the external and internal phases. As shown in Figure 4 Figure 3A, when the external phase flow rate is reduced from 7200 μl / h to 2400 μl / h, the average particle size of the microcapsules significantly increases from 565.24 μm to 924.27 μm Figure 4 A). In which, Comparative Example 3 forms smaller microcapsules due to the too large external phase flow rate, which however leads to a large-scale fusion of the internal and external phases. In addition, Comparative Example 4 forms oval microcapsules due to the too small external phase flow rate. In contrast, the microcapsules of Example 1 are more monodisperse and have a more obvious core-shell structure. Because the shape of the core of the core-shell microcapsule is different, the size of the core cannot be directly measured by the diameter. However, under the same flow rate, we believe that the volume of the core is similar, so under different internal phase flow rates, the internal phase flow rate is optimized by fixing the amount of the internal phase, and the number of the produced core-shell microcapsules. When the internal phase flow rate is increased from 200 μl / h to 600 μl / h, the number of the produced microcapsules is significantly reduced from 1537 to 511, which reflects the increase of the volume of the internal phase Figure 4 B). In which, Comparative Example 5 leads to a smaller internal phase volume of the core-shell microcapsule due to the too small internal phase flow rate, which inevitably leads to a decrease in the loading capacity of the core-shell microcapsule, while the too large internal phase flow rate in Comparative Example 6 leads to the disappearance of the core-shell structure, which is not conducive to the protection of the probiotics by the subsequent microcapsules.

[0141] In summary, the present application demonstrates the successful preparation of the core-shell microcapsule with a multi-layer structure in the present application by comparing Example 1 and Comparative Examples 1-2. At the same time, by comparing Example 1 and Comparative Examples 3-4, and by comparing Example 1 and Comparative Examples 5-6, the influence of the flow rates of the external and internal phases on the average particle size and volume of the final microcapsules is found, and in combination with the purpose of loading bacteria in the present application, the flow rates of the external and internal phases are preferably the flow rates in Example 1, i.e. the internal phase flow rate is 500 μL / h, and the external phase flow rate is 3600 μL / h.

[0142] Example 2

[0143] This embodiment is the process for preparing the bacteria-loaded core-shell microcapsule, and the preparation process of the core-shell microcapsule is referred to the steps of Example 1, wherein other preparations are unchanged, and only the preparation of the internal phase is changed, i.e. the process of step (3) in Example 1 is changed to the following two steps:

[0144] 1. Preparation of Lactobacillus plantarum powder: Lactobacillus plantarum was inoculated in MRS broth and incubated at 37°C for 16 h to activate the probiotic bacteria. After two subcultures, the probiotic bacteria were collected by centrifugation at 8000 rpm for 8 min at 4°C. The probiotic bacteria precipitate was washed twice with sterile physiological saline and then 10 times the volume of a freeze-drying protective solution containing skimmed milk powder (7.5 wt%) and inulin (7.5 wt%) was added. The Lactobacillus plantarum powder was obtained by freeze-drying and had a viable count of 10 log CFU / g. The Lactobacillus plantarum powder was added to corn oil at a ratio of 1 : 15 (g / mL) and vortexed to obtain a homogeneous suspension in corn oil.

[0145] 2. Preparation of core material for the core-shell microcapsule: Salmon protein powder was dissolved in sterile water and stirred to obtain a 4 wt% salmon protein solution. The 4 wt% salmon protein solution was mixed with a 2 wt% sodium alginate solution at a volume ratio of 1 : 1 and homogenized at 8000 rpm for 30 s to obtain a mixed solution. The pH of the mixed solution was then adjusted to 7, and the Lactobacillus plantarum corn oil was added to the mixed solution (volume ratio of oil phase to water phase 1 : 1, v / v). The mixture was then homogenized at 12000 rpm for 2 min to obtain a stable Pickering emulsion, i.e. the core material for the core-shell microcapsule.

[0146] The remaining steps were prepared according to the preparation in Example 1, and then the probiotic bacteria-loaded core-shell microcapsule was prepared according to step (5).

[0147] Comparative Example 7

[0148] This comparative example is the preparation of a bacteria-loaded single-shell microcapsule. The outer phase oil phase was unchanged, and the preparation of the intermediate phase was as follows:

[0149] Preparation of probiotic bacteria-loaded single-shell microcapsule: Lactobacillus plantarum was inoculated in MRS broth and incubated at 37°C for 16 h to activate the probiotic bacteria. After two subcultures, the probiotic bacteria were collected by centrifugation at 8000 rpm for 8 min at 4°C. The probiotic bacteria precipitate was washed twice with sterile physiological saline and then 10 times the volume of a freeze-drying protective solution containing skimmed milk powder (7.5 wt%) and inulin (7.5 wt%) was added. The Lactobacillus plantarum powder was obtained by freeze-drying and had a viable count of 10 log CFU / g. The Lactobacillus plantarum powder was added to corn oil at a ratio of 1 : 15 (g / mL) and vortexed to obtain a homogeneous suspension in corn oil.

[0150] Then, the probiotic bacteria-loaded single-shell microcapsule was prepared according to the relevant preparation parameters described in Comparative Example 1.

[0151] Comparative Example 8

[0152] This comparative example is the preparation of bacterial-loaded mononuclear microcapsules. Referring to Comparative Example 2, the external oil phase remains unchanged, while the internal phase is operated as follows:

[0153] 1. Preparation of Lactobacillus plantarum powder and oil: Lactobacillus plantarum was inoculated into MRS broth and incubated at 37°C for 16 h to activate the probiotics. After two subcultures, the probiotic precipitate was collected by centrifugation at 8000 rpm for 8 min at 4°C. The probiotic precipitate was washed twice with sterile physiological saline, and then 10 times the volume of a freeze-drying protectant containing skim milk powder (7.5 wt%) and inulin (7.5 wt%) was added. The mixture was then freeze-dried to obtain Lactobacillus plantarum powder with an activity level of up to 10 log CFU / g. The Lactobacillus plantarum powder was then added to corn oil at a ratio of 1:15 (w / v) and vortexed to ensure uniform suspension in the corn oil, thus obtaining Lactobacillus plantarum corn oil.

[0154] 2. Preparation of core-shell microcapsule core material: Salmon protein powder was dissolved in sterile water and stirred to prepare a 4wt% salmon protein solution. The 4wt% salmon protein solution was mixed with 2wt% sodium alginate solution at a volume ratio of 1:1 and homogenized at 8000 rpm for 30 s to prepare a mixed solution. The pH of the mixed solution was then adjusted to 7. Then, Lactobacillus plantarum corn oil was added to the mixed solution (the volume ratio of oil phase to water phase was 1:1, v / v). After that, it was homogenized at 12000 rpm for 2 min to prepare a stable Pickering emulsion, which is the core-shell microcapsule core material (inner phase).

[0155] Then, according to the relevant preparation parameters described in Comparative Example 2, probiotic-loaded single-nuclear microcapsules were prepared.

[0156] Using free probiotics as a control group, the survival rate of probiotics encapsulated in core-shell microcapsules (Example 2), single-shell microcapsules (Comparative Example 3), and single-core microcapsules (Comparative Example 4) after gastrointestinal digestion was determined. Figure 4 As shown in Figure a, the free probiotics were completely inactivated after the simulated digestion. This is because the extremely low gastric pH damages the cell membrane of the probiotics, thereby destroying their DNA and proteins. Microencapsulation technology can significantly enhance the activity of probiotics after simulated digestion. Specifically, the activity of *Lactobacillus plantarum* encapsulated in core-shell microcapsules, single-shell microcapsules, and single-core microcapsules decreased from the initial 8.48, 8.12, and 8.19 log CFU / g to 6.84, 6.20, and 5.70 log CFU / g, respectively. Compared to Comparative Examples 3 and 4, Example 2, by immobilizing the probiotics in the core emulsion, maximally isolates them from the harsh conditions of the gastrointestinal tract, thus delaying the time the probiotics come into contact with digestive juices. Further imaging of the microcapsules at different time points during simulated digestion was conducted to investigate the protective mechanism of the microcapsules on the probiotics. Figure 4As shown in Figure b, it was observed that all three groups of microcapsules could maintain their initial shape after the end of the simulated gastric fluid, due to the pH-responsive ability of calcium alginate, in which the carboxyl groups of calcium alginate were in a non-ionized state in a low pH environment. The morphologies of the single-layer microcapsules and the core-shell microcapsules showed different trends in the simulated intestinal fluid. The single-layer microcapsules first swelled rapidly and then gradually dissolved, because in a neutral environment, the carboxyl groups of calcium alginate ionized, which increased the electrostatic repulsion between them, thereby accelerating the disintegration. The core-shell microcapsules first dissolved the shell layer, and then the core layer swelled. At the end of the simulated intestinal fluid, they still maintained a certain structure. This was due to the dual effects of the cross-linking of the shell layer calcium alginate and the kelp nanocellulose and the filling of the oil droplets in the core layer, which significantly increased the disintegration time of the core-shell microcapsules. Therefore, it can be inferred that the core-shell microcapsules can hinder the direct contact of probiotics with gastric acid and bile salts by relieving the disintegration time of the microcapsules, thereby significantly improving the survival rate of probiotics.

[0157] Table 1 Live cell counts of the microencapsulated probiotics during simulated gastrointestinal digestion

[0158]

[0159] The survival of the probiotics encapsulated in the core-shell microcapsules (Example 2), single-shell microcapsules (Comparative Example 3), and single-core microcapsules (Comparative Example 4) was determined after 4 weeks of storage in fruit juice at 4°C and 25°C, respectively, with free probiotics as the control group. The specific results are shown in Table 2 and Table 3. Figure 5 At 25°C storage, the survival rate of probiotics in all cases was lower than at 4°C storage, indicating that Lactobacillus plantarum was sensitive to storage temperature. Because higher temperatures often lead to higher metabolic activity. In addition, it was observed that the probiotics in all treatment groups showed faster loss of activity in the first week, and the loss of activity became slow in the subsequent storage period, which can be attributed to an acid adaptation mechanism. After the end of storage, whether at 4°C or at 25°C, the single-core and single-shell microencapsulated probiotics, and the core-shell microencapsulated probiotics, all significantly improved the activity of the probiotics compared to free probiotics. This indicates that multi-layer microencapsulation can better isolate antibacterial agents such as citric acid and vitamin C in orange juice to maintain cell integrity.

[0160] Table 2 Live cell counts during 4 weeks of storage in fruit juice at 25°C

[0161]

[0162] Table 3 Live cell counts during 4 weeks of storage in fruit juice at 4°C

[0163]

[0164]

[0165] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing a chitosan-coated microcapsule, characterized by, The method comprises the following steps: (1) Preparation of hydrogel precursor solution: CaCl2 solution is mixed with Na2-EDTA solution and the pH is adjusted to neutral with NaOH aqueous solution, followed by stirring, and then sodium alginate is added and uniformly mixed to prepare the hydrogel precursor solution; (2) Preparation of Lactobacillus plantarum powder oil: the probiotic bacteria are inoculated in MRS broth and incubated at 35-38°C for 15-18 h to activate the probiotic bacteria, after two subculture, the probiotic bacteria precipitate is collected by centrifugation, and then the probiotic bacteria precipitate is washed twice with sterile normal saline, then a freeze-drying protective solution is added, and then freeze-drying is performed to obtain probiotic bacteria powder, and then the probiotic bacteria powder is added to vegetable oil to prepare probiotic bacteria vegetable oil; (3) Preparation of core-shell microcapsule core material: protein is stirred in water to prepare a protein solution, the protein solution is mixed with a sodium alginate solution to prepare a mixed solution, and then the pH value of the mixed solution is adjusted to neutral, and then the probiotic bacteria vegetable oil is added to the mixed solution and uniformly mixed to obtain the core-shell microcapsule core material; (4) Preparation of core-shell microcapsule shell material: nanocellulose is dissolved in water to obtain a nanocellulose solution, and then the nanocellulose solution is mixed with the hydrogel precursor solution prepared in step (1) to obtain the core-shell microcapsule shell material; The nanocellulose is kelp nanocellulose; (5) Production of core-shell microcapsules: Tween 80, acetic acid and vegetable oil are mixed to prepare an external oil phase, and then the core-shell microcapsule core material prepared in step (3) is used as an inner phase, and the core-shell microcapsule shell material prepared in step (4) is used as an intermediate phase, which are pumped into a microfluidic chip respectively, droplets of the inner gel are formed in the chip, and then collected in a CaCl2 solution, and then centrifuged to obtain the core-shell microcapsules; the flow rate of the inner phase is 500 μL / h; the flow rate of the intermediate phase is 800 μL / h; and the flow rate of the external phase is 3000-4000 μL / h.

2. The method as claimed in claim 1, characterized in that In step (1), the concentration of the CaCl2 solution is 50-120 mM, the concentration of the Na2-EDTA solution is 50-120 mM, and the volume ratio of the CaCl2 solution to the Na2-EDTA solution is 1:0.8-1.2; and the mass fraction of sodium alginate in the hydrogel precursor solution is 0.5-4%.

3. The method of claim 1, wherein, In step (2), the probiotic bacteria include one or more of Bifidobacterium, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactobacillus fermentum and Lactobacillus helveticus; and the vegetable oil is one or more of corn oil, soybean oil, rapeseed oil, peanut oil, algal oil and palm oil.

4. The method as claimed in claim 1, characterized in that In step (2), the freeze-drying protective solution is a mixed aqueous solution of a sugar and a protein; the sugar is inulin, and the protein is skimmed milk powder; the concentration of the inulin is 5-10 wt%, and the concentration of the skimmed milk powder is 5-15 wt%.

5. The method as claimed in claim 1, wherein, In step (3), the protein includes one or more of salmon protein and cod protein; the mass concentration of the protein in the protein solution is 1-5%; the mass concentration of sodium alginate in the sodium alginate solution is 0.5-4%; and the volume ratio of the protein solution to the sodium alginate solution is 1:0.8-1.

2.

6. The method of claim 1, wherein, The mass concentration of nanocellulose in the nanocellulose solution in step (4) is 0.1-0.5%; the volume ratio of the nanocellulose solution to the hydrogel precursor solution is 1:0.8-1.

2.

7. The microcapsules with bacterial nuclei prepared by the method according to any one of claims 1-6.

8. The use of the microcapsules with bacterial nuclei according to claim 7 in the preparation of food, medicine and health products.

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